1. Field of the Invention
This invention relates to chemical sensors, more particularly, to a method of making a indicator attached to a macromolecule immobilized in sol-gel glass. The immobilized macromolecule is entangled in the sol-gel so that even temperatures experienced during autoclaving do not cause the macromolecule or the indicator to leach or otherwise separate from the sol-gel glass.
2. Description of Related Art
Chemical sensors, in order to be reliable, reproducible and practical, usually require that whatever chemistry is incorporated into the sensor does not leach out into the surrounding matrix. Leaching can degrade the sensor's performance as well as contaminate the sample. Typically this problem has been addressed by covalently bonding molecules of interest to a solid support, or creating a polymer in which the molecule of interest is incorporated into the polymer matrix during the polymerization process. In the case of optically based sensors, the optical properties of the support are also important.
Sol-gel glasses have been used as a basis for chemical sensors. Sol-gel glass is an optically transparent amorphous silica or silicate material produced by forming interconnections in a network of colloidal, submicrometer particles under increasing viscosity until the network becomes completely rigid, with about one-half the density of glass. (For a comprehensive text, readers may refer to Sol Gel Science by Jeffrey C. Brinker and George W. Scherer, Academic Press, Inc., San Diego, 1990.)
The sol-gel process comprises hydrolysis and condensation of starting monomers to produce a colloidal suspension (the "sol"), gelation (to form a wet network of porous metal oxide), and drying (and shrinking) to form the "xerogel" (i.e. dry gel); final sintering (optional) at elevated temperature densities the xerogel into pore-free glass. A general discussion of sol-gel porous glass technology can be found in "Diagnostic Applications of Organically Doped Sol-Gel Porous Glass", O Lev, Analusis, 1992, v20, N9 (Nov), p543-553. Typically, the sol-gel process begins with soluble precursors. Usually these are metal-organic derivatives such as tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS), which react with water to form extremely small colloidal structures that comprise the sol. While mixing the liquid precursor with the water, a hydrolysis reaction occurs. The hydrated silica immediately interacts in a condensation reaction forming Si--O--Si bonds.
Linkage of additional Si--OH tetrahedra occurs as a polycondensation reaction, eventually resulting in a SiO.sub.2 network. Hydrolysis and polycondensation reactions initiate at numerous sites within the TMOS or TEOS aqueous solution as mixing occurs. When sufficient interconnected Si--O--Si bonds are formed in a region they respond cooperatively as a colloidal (submicron) particle, or pre-network. The suspension of these colloidal particles in their parent liquid is termed a sol. The sol still behaves as a low-viscosity liquid and can be cast into a mold.
After casting into a mold, gelation occurs: the colloidal particles link together to become a three-dimensional network. When gelation occurs, viscosity increases sharply and a solid results. Aging of a gel involves keeping the gel immersed for some period of time (hours to days), during which time the gel decreases in porosity and develops the strength necessary to resist cracking during drying (curing).
During drying, the pore liquid is removed and evaporation is controlled to avoid stress cracks. The density of dried gels ranges from as low as 5% of the density of a melt-derived material to as much as 60% of theoretical density. Low-density gels are called aerogels; high density gels are xerogels.
The sol-gel glass is optically transparent but contains a large fraction of interconnected pores. Small indicator molecules of various kinds can be incorporated into the porous matrix during the formation of the sol-gel. Because the molecules are small, they tend to diffuse out of the glass, particularly at elevated temperatures.
The sol-gel technique has also been shown as a way to immobilize enzymes. A biosensor can be made by entrapping the enzyme in the porous matrix during the formation of the xerogel. The enzyme remains active and resistant to leaching, being physically trapped or entangled in the three-dimensional silica structure created during the sol-gel process. Enzymes are limited by a number of characteristics, including temperature and solvent sensitivity, because such sensitivities restrict the applications of enzyme-based sensors.
Leaching has been a persistent problem in chemical sensors irrespective of the formation technique. Unless prevented, molecules can (and generally do) leach off the matrix support. Covalent attachment of indicator molecules has been used to prevent indicator leaching, as well as to prevent the changes resulting from leaching, such as changes in the surface concentration of indicator and the contamination of the surrounding liquid. Typically, one derivatizes a surface such that pendant groups can then be reacted and bonded covalently with the indicator molecules. For example, aminopropyltriethoxylane can be reacted with a silica surface to form "amino-propyl silica." The pendant amino groups can be reacted with a carboxyl group on the indicator molecule to form a stable amide bond.
Sensors based on the incorporation of small indicator molecules into a sol-gel glass can also be unstable due to the leaching of the indicator. Leaching is especially problematic if the sensor is exposed to high temperatures. Smaller pore sizes slow the leaching process but, at least in block sensors, have the undesirable characteristic of retarding the response time of the sensor, since response time is a function of the rate of diffusion. Sol-gels made as thin films, however have small pores and faster response times because diffusion remains rapid.
Enzymes, by virtue of being entangled or otherwise immobilized in the sol-gel, do not present the same leaching problem as small indicator molecules but, rather, pose other difficulties. Although entangled enzymes offer the possibility of very sensitive and specific analyses, enzymes are not suited for all applications. Enzymes have limited lifetimes, are sensitive to solvents, and are not stable at the temperatures required for sterilization.